Introduction
Cholera can be defined as a sudden onset of watery diarrhea often accompanied by vomiting and resulting in hypovolemic shock and acidosis. Cholera is caused by certain members of the species Vibrio cholerae which reside in the small intestine and secrete cholera toxins.
Vibrio cholerae strains are natural inhabitants of brackish water and estuarine systems where they may constitute the normal microflora of zooplankton and shellfish. Vibrio cholerae is the type species of the genus Vibrio that belongs to the family Vibrionaceae. Vibrio cholerae are facultative anaerobe, highly motile and slightly curved non-sporing, gram negative rods measuring 1.4 to 2.6 μm in length. Vibrio cholerae grows optimally in the presence of 5-15 mM sodium ions (Na+) and can tolerate high alkalinity (˜pH 10) (Reidl et al., 2002; Faruque et al., 1998; Parsi, 2001; Cooper, 2001; Mooi et al., 1997).
Out of 200 vibrio serovars, only Vibrio cholerae O1 and O139 have been implicated as causative agent of epidemic and pandemic cholera. It is known that the 5th and 6th pandemics were caused by V. cholerae O1 of classical biotype, but the nature of the strains causing the first four pandemics is not known. Although it is generally believed that the first three pandemics were also caused by classical biotypes. Prior to 7th pandemic, the El Tor biotype was thought not to cause cholera, since it was associated with only mild diarrhea. However, it was later confirmed that both, the classical and El Tor biotypes have potential to cause serious cholera.
Non-O1 serotypes were never associated with cholera epidemics, although they have caused local outbreaks. Until mid 1992, it was generally considered that the ongoing seventh pandemic is caused by Vibrio cholerae O1. Beginning in October 1992, cases in Madras, India were noted which were associated with a Vibrio cholerae strain that did not agglutinate with O1 antisera. Similar cases were subsequently noted in Madurai, Vellore, Calcutta, and in Southern Bangladesh. In the following months, this new strain spread in epidemic form across Bangladesh, with over 100,000 cases reported by the end of March 1993. Cases were also reported from Thailand, Malaysia, Pakistan, and Nepal. For a time being, this new strain replaced V. cholerae O1 strain as the predominant cause of cholera in Southeast Asia. It was also isolated from travelers in California, Estonia, Germany, Singapore, and HongKong.
This new strain was later designated as Vibrio cholerae O139 synonym Bengal. V. cholerae O139 is closely related to the Asian V. cholerae O1 El Tor strain; however, it has certain distinctive features. Features that are common between O139 and El Tor are high level of polymyxin B resistance, capability of agglutinating chicken erythrocytes and resistance to infection by Mukhejee's phage. V. cholerae O139 does not agglutinate with monoclonal or polyclonal antisera directed against the O1 antigen. These strains appear to lack portion of at least two genes within the O1 biosynthetic gene cluster (Vcrfb) and therefore do not express native O antigen. Analysis of serogroup O1 and O139 isolates has revealed marked differences in their Lipopolysaccharide (LPS). O139 strain appears to have a modified core structure and is usually referred to as a semi-rough LPS and not a smooth LPS like in the O1 strain. Due to the presence of the LPS capsule, the O139 strain is resistant to killing by healthy human serum and has increased potential for invasive disease among persons infected with this organism.
Cholera has re-emerged as a major infectious disease in the recent past, with a global increase in its incidence. People in fifty eight countries across the globe are suffering from cholera pandemic. An estimated of 184,311 cases were reported to WHO with 2,728 death in year 2001 alone; however, officially notified cases do not reflect the overall burden of disease. Most under developing countries deliberately underreport cholera cases due to the fear of travel and trade related sanctions. A careful estimate suggests that some 5-7 million cholera cases occur annually with around 100-120,000 deaths each year.
Despite great efforts made by several countries, cholera is again on the rise. While the disease no longer poses a threat to countries with minimum standards of healthy living conditions, it remains a challenge to countries where access to safe drinking water and adequate sanitation cannot be ensured for all. Almost every developing country is facing either a cholera outbreak or the threat of an epidemic.
Cholera patients who receive adequate treatment mostly always recover rapidly. Treatment of cholera consists essentially of replacing fluid and electrolytes either intravenously or orally. Antimicrobial therapy serves as an important adjunctive therapy.
Due to the practical limitations of the implication of various preventive measures in cholera endemic areas, mass vaccination of the population at risk offers a cheap prophylactic alternative. Attempts of developing cholera vaccine had started from 1884 but with limited success; however, researchers have been able to gather invaluable data from all those earlier attempts.
Cholera Vaccines
The idea that an initial clinical infection gives rise to high level of enduring protection was not appreciated until the pioneering work of Cash et al in 1974 demonstrated that an initial infection with classical V. cholerae O1 Inaba, provides solid protection for at least 1 year against re-challenge with the homologous organism. These observations were further expanded by Levine between 1979 and 1983, who demonstrated that an initial clinical infection with the classical biotype V. cholerae O1 provides 100% protection against illness following re-challenge with the classical biotype organisms of either serotype for at least 3 years (Levine et al., 1993). However, the same authors reported a 90% protection after the infection with V. cholerae O1 El Tor. The authors also noted that no vibrios could be recovered from the excreta of the volunteers who were re-challenged with the classical biotype. In contrast, the authors were able to recover vibrios from co-procultures of approximately one third of the volunteers who were challenged and subsequently re-challenged with El Tor vibrios (Levine et al., 1993 and 1995).
Volunteers orally fed with as little as 0.5 microgram of purified cholera toxin suffer with profuse diarrhea, whereas immunized volunteers while fed with purified cholera toxin don't (Levine et al., 1993 and 1995). This suggests that the presence of anti-cholera toxin antibodies on the surface of the mucosa in the small intestine is capable of neutralizing the cholera toxin thus prevents the onset of the watery diarrhea. On the other hand, immunized volunteers while challenged with homologous virulent V. cholerae, shed very little if any virulent vibrios in co-proculture (Levine et al., 1995 and 1993). This clearly suggests that antibodies are present on the surface of the mucosa in the small intestine that neutralize the virulent vibrios thus preventing them from colonizing or secreting cholera toxin in the lumen (Levine et al., 1995 and 1993). Considerable direct and indirect evidence from epidemiological studies, involving human volunteers as well as animal studies points to the existence of both, anti cholera toxin and antibacterial immunity (Bondre et al., 1997; Pierce et al., 1987). These studies have also revealed that a better protection is achieved while using both, the toxin and the somatic antigens that involve a synergistic interplay between antitoxic and antibacterial immune mechanism (Ryan et al., 2000).
Parenteral Cholera Vaccines
Until it was found in mid 1970s that V. cholerae is a non-invasive organism, almost all the vaccines developed against cholera were given parenterally. Various type of parenteral vaccines developed include killed whole cell vaccines, toxoid vaccines, and combined whole cell toxoid vaccines.
Killed Whole Cell Parenteral Vaccines
Since 1884, killed whole cell vaccines made with V. cholerae O1 have been utilized as parenteral vaccines; however, it was not until early 1960s, that well designed randomized controlled trials to assess the efficacy of these vaccines have been carried out. However, it was found that these vaccines provided with only short term protection and that the protection was also age related (Ryan et al., 2000). Adults afforded better protection than the young children, suggesting that the vaccine worked best in immunologically primed populations by boosting underlying immunity (Ryan et al., 2000). In a few trials, killed whole cell vaccine was administered with adjuvants in hope to enhance the immune response, however, this led to serious local reactions and further trials were abandoned (Ryan et al., 2000; Kalambaheti et al., 1998).
Toxoid Parenteral Vaccines
A number of toxoid vaccines have been developed to be administered parenterally and were intended to protect the vaccinee by eliciting antitoxic immunity.
These include formaldehyde cholera toxoid, gluteraldehyde cholera toxoid, procholeragenoid, and B subunit toxoid vaccine (Ryan et al., 2000; Levine et al., 1995 and 1993).
Formaldehyde Cholera Toxoid Parenteral Vaccine
It was found that the treatment of purified cholera toxin with formaldehyde could eliminate its toxicity without compromising its ability to stimulate anti-toxin antibodies (Ryan et al., 2000). A prototype vaccine candidate was prepared with alum adjuvant and administered to volunteers in Bangladesh. The vaccine prototype elicited significant levels of antitoxin IgG without severe local reaction at the site of injection. However, field trials to assess the efficacy of this vaccine were never initiated (Ryan et al., 2000; Levine et al., 1995 and 1993). A modification of this vaccine prototype was prepared using formalin and glycine and termed lot 11. A large scale field trial was conducted in Philippines but no beneficial effects were detected, therefore, further trials were abandoned (Ryan et al., 2000; Levine et al., 1995 and 1993).
Gluteraldehyde Cholera Toxoid Parenteral Vaccine
A gluteraldehyde treated cholera toxoid vaccine was prepared in the hope of getting a somatic antigen free toxoid vaccine. Field trials conducted in Bangladesh in parallel to a killed whole cell vaccine and a subunit vaccine revealed little protection afforded by this vaccine candidate. Accordingly, no further trials were conducted (Ryan et al., 2000; Levine et al., 1995 and 1993).
Procholeragenoid Parenteral Vaccine
Purified cholera toxin heated at 65° C. for 5 minutes followed by formaldehyde treatment yields procholeragenoid, a high molecular weight toxoid that retains its immunogenic potential without any toxicity. Studies conducted in rabbit model have revealed high serum antitoxic IgG titers comparable to titers obtained with untreated cholera toxin. However, this vaccine prototype has never been tried in humans (Levine et al., 1993; Germanier et al., 1976 & 1977).
B Subunit Toxoid Parenteral Vaccine
Biological activity of whole cholera toxin is due to it's A subunit, whereas B subunit is potentially immunogenic. Since B subunit is highly immunogenic and has no toxic activity, it represents an attractive immunogen to stimulate antitoxic immunity. While administered in human volunteers, the B subunit toxoid vaccine exhibited high titers of antitoxic antibodies in the serum; however, the immunity was higher in immunologically primed populations (Ryan et al., 2000; Levine et al., 1993; Sack et al., 1991).
Bacterial Subunit Parenteral Vaccines
Bacterial subunits such as LPS have also been administered parenterally to elicit the immune response. One such vaccine prototype was prepared using purified Ogawa and Inaba LPS-protein extract. The efficacy of this prototype was evaluated in Bangladesh where Ogawa LPS vaccine yielded almost similar efficacy as killed whole cell vaccine. Similarly in another field trial, Inaba LPS vaccine provided a level of protection as high as that conferred by the Inaba whole cell vaccine (Ryan et al., 2000; Levine et al., 1993; Sack et al., 1991).
Combined Whole Cell Toxoid Parenteral Vaccines
In the 1970s, Welcome Research Laboratories prepared a combined whole cell toxoid vaccine administered parenterally with alum adjuvant. The combination vaccine stimulated excellent serum vibriocidal and antitoxic responses; however, this vaccine prototype was never evaluated for its efficacy in large scale controlled field trials (Ryan et al., 2000; Levine et al., 1993; Sack et al., 1991).
Oral Cholera Vaccines
Until 1970, the majority of cholera vaccines were developed for parenteral administration, nevertheless, few cholera vaccines were also developed for oral administration. However, earlier trials with oral cholera vaccines were empirical and not based on immunological principles. From the mid 1970s, researchers started appreciating the role of oral immunization based on the fact that natural infection with V. cholerae elicits high level of protection that lasts for at least 3 years.
Killed Whole Cell Oral Vaccines
Modern history of orally administrable killed whole cell vaccines starts in 1962 when Freter et al., 1962 reported 77% efficacy of this whole cell vaccine in North American volunteers. Cash in 1974, evaluated the efficacy of a whole cell vaccine composed of 1.6×1010 killed Ogawa and Inaba O1 vibrios in North American volunteers with an efficacy of 61%. A Swedish killed whole cell vaccine composed of 2×1011 heat and formalin killed Ogawa and Inaba O1 vibrios have also been extensively evaluated in North American and Swedish adults and in Bangladeshi adults and children. The vaccine did not cause any side effects and a significant rise in serum vibriocidal antibodies was seen in 80% of volunteers. Clemens has reported a 52% vaccine efficacy of a whole cell killed vaccine for at least 36 months in a controlled field trial in Bangladesh (Clemens et al., 1988 & 1990).
Bacterial Fraction Oral Vaccine
A bacterial fraction vaccine has been prepared by treating the outer membrane of V. cholerae with trichloroacetic acid. The antigenic complex thus extracted was referred to as CH1+2. This vaccine candidate was evaluated in a field trial in Zaire with a total of 18,623 individuals vaccinated. The vaccine was found to elicit good immune response. This vaccine has not been evaluated for efficacy in other field trials (Fournier 1998).
Gluteraldehyde Cholera Toxoid Oral Vaccine
North American volunteers were orally vaccinated with three doses of 2 mg of gluteraldehyde treated toxoid 1 month apart or three 8.0 mg oral doses with NaHCO3 at 1 month intervals. No adverse effects were observed, however, when experimental challenge studies were carried out to assess the protective efficacy, no significant protection was observed (Ryan et al., 2000; Levine et al., 1993).
B Subunit Toxoid Oral Vaccine
Swedish and Bangladeshi volunteers were vaccinated orally with the B subunit of highly purified cholera toxin with or without sodium bicarbonate (NaHCO3).The vaccine candidate caused no reactogenic side effects and stimulated high titers of antitoxin antibodies in serum, saliva and breast milk. Administration with NaHCO3 or other gastric acidity neutralizing buffer enhanced the immune response. Large-scale field trials with the B subunit alone were not conducted; however, it has been evaluated extensively in combination with whole cell killed oral vaccine (Ryan et al., 2000; Levine et al., 1993).
Combination Oral Vaccines
Three different killed whole cell vaccines have been tried in combination with toxoid as oral vaccines. These include (i) gluteraldehyde cholera toxoid combined with alcohol killed O1 vibrios, (ii) procholeragenoid combined with heat and formalin killed O1 vibrios and (iii) B subunit combined with heat or formaldehyde inactivated O1 vibrios. All three combinations were well tolerated by the volunteers. However, the B subunit combined with killed whole cells gave the best immune response. Significant rises in serum vibriocidal antibodies were seen in 89% of the vaccinees. In another study, the B subunit/killed whole cell combination vaccine was compared with oral whole cell vaccine alone. A total of 63,498 individuals were recruited for a randomized, double blind placebo controlled trial in Bangladesh. During the first six months of surveillance the level of protection provided by the combination vaccine was significantly higher than that of whole cell vaccine alone (85% vs 65%). After 12 months of surveillance, the level of protection was 62% vs 53% for combination vaccine and whole cell killed vaccine respectively. During the first six months, the combination vaccine protection was similar in young children as well as in older children and adults. However after six months, the level of protection dropped appreciably in children less than six years old (Ryan et al., 2000; Taylor et al., 1999; Concha et al, 1995; Jertborn et al., 1996).
Despite the significant advantages of this combination vaccine, it nonetheless had drawbacks that limited its use. First it has transient protection in the young children, the group which is most susceptible to cholera infection. Second the cost involved in the preparation of the vaccine. Third is the requirement for multiple (at least two) spaced doses to prime and boost the immune response.
Live Oral Vaccines
For many years, some investigators have favored the concept of live oral vaccine as it best mimics the natural infection. It is also well established that only the natural infection provides with best protection for at least three years. Many live oral vaccines have been developed and evaluated in human volunteers for their efficacy.
Environmental Strains as Live Oral Vaccines
A number of non-enterotoxigenic V. cholerae O1 strains have been isolated from the environment and have been evaluated in volunteers for their efficacy to elicit immunity. The results were generally disappointing as most of these environmental strains were poor colonizers of the small intestine and therefore were unable to evoke immune response.
Chemically Mutated Attenuated Strains as Live Oral Vaccines
Before the advent of modern tools to engineer the DNA, mutations were generally made by exposing the DNA to mutagenizing chemicals such as nitrosoguanidine. Two such strains of V. cholerae attenuated by mutagenizing agent nitrosoguanidine are M13 and Texas Star-SR. M13 was prepared from pathogenic classical Inaba 569B. When given orally to the volunteers, M13 did not cause any diarrhea, and elicited moderate but significant level of protection against challenge with pathogenic V. cholerae O1.
Texas Star-SR was an A−B+ mutant derived from El Tor Ogawa strain 3083. Texas Star-SR was mildly reactogenic in volunteers; however, it provided moderate but significant protection against challenge with either serotype of El Tor. Texas Star-SR suffered from certain inherent drawbacks, i.e. mutagenesis with nitrosoguanidine is known to induce multiple mutations, and the precise genetic lesion presumed responsible for the attenuation was unknown. Therefore, there always remained the theoretical possibility that it could revert to virulence.
Attenuated Mutants Prepared by Recombinant DNA Technique
It has generally been accepted that diarrhea is caused by the cholera toxin. And therefore, mutating or deleting the whole gene coding for cholera toxin or mutating the gene coding for the biologically active A subunit would be sufficient to make a vaccine strain non-toxic. Based on this hypothesis, a number of vaccine strains have been constructed such as JBK70, CVD101, CVD103, CVD104, CVD105, O395-N1.
JBK70 was a A−B− strain originally derived from the toxigenic N16961 strain. In human volunteers it did not cause severe diarrhea and elicited high titers of vibriocidal antibodies. When the vaccines were challenged with the virulent N16961, only 1 out of 10 vaccinees developed severe diarrhea as compared to 7 out of 8 non-vaccinated individuals. Thus 89% of protection was achieved. Further studies with JBK70 were halted due to mild diarrhea experienced by most of the volunteers (Ryan et al., 2000; Levine et al., 1993).
Since JBK70 was mildly reactogenic, in order to ascertain whether this reactogenicity was associated with the El Tor strain, a vaccine strain CVD101 was developed (Ryan et al., 2000; Levine et al., 1993). CVD101 was a A−B+ strain derived from classical Ogawa 395 strain (Ryan et al., 2000; Levine et al., 1993). When orally administered to the volunteers, 40-60% of volunteers still developed mild diarrhea. However, both the antitoxin and vibriocidal titers were comparable to that achieved with the toxigenic parent strains (Ryan et al., 2000; Levine et al., 1993). Although able to evoke very good immune response, both the JBK70 and CVD101 caused mild diarrhea in the volunteers. This indicated that V. cholerae of either serotype secrete some toxin other than cholera toxin that is responsible for the residual reactogenicity. Suggested candidates for this effect were the hemolysin/cytotoxin, cytotoxin/protease, or shiga like toxin (Ryan et al., 2000; Levine et al., 1993).
In order to investigate this hypothesis, gene coding for hemolysin was deleted from JBK70 and CVD101 resulting in strains CVD104 and CVD105 respectively. While orally fed, volunteers still suffered with mild diarrhea. All vaccinees shed the vaccine strain and the immune response was satisfactory (Ryan et al., 2000; Levine et al., 1993; Kaper et al., 1990).
In an effort to develop a non-toxigenic vaccine strain, an A−B+ strain was derived from the classical Inaba 569B that does not elaborate Shiga like toxin and was designated as CVD103 (Kaper et al., 1990). CVD103 did not cause diarrhea when given to the volunteers and it elicited good vibriocidal as well as antitoxic immunity after a single dose. At high doses of 1×108 cells, only 5 out of 46 volunteers developed mild diarrhea and out of 5, only one volunteer had stool volume that exceeded 400 ml. In contrast to JBK70, CVD101 and O395-N1, that frequently resulted in malaise, anorexia, and abdominal cramps, these symptoms were never recorded among volunteers who received CVD103 (Kaper et al., 1990). In order to distinguish the mutant from the wild type, a mercury resistant gene was cloned onto the hlyA locus of CVD103, thus obtained CVD103-HgR. An initial study with CVD103-HgR was conducted in 18 North American volunteers. No one experienced adverse reactions and less organisms were recovered from the co-procultures as compared to CVD103. Nevertheless, CVD103-HgR colonized the small intestine and elicited the vibriocidal antibodies and antitoxic antibodies similar to those who were vaccinated with parent CVD103 (Cryz et al., 1995). Based on the encouraging studies done with North American volunteers, studies were initiated with volunteers in several countries in Africa, Asia and Latin America. Experimental challenge studies in volunteers demonstrated protection as early as 1 week after vaccination. A high level of protection (>90%) was conferred against moderate and severe cholera caused by challenge with V. cholerae O1 of either El Tor or classical biotype. The overall protective effect against El Tor cholera of any severity was 80% (Ryan et al., 2000).
A few limitations associated with CVD103-HgR include the lack of evidence whether this vaccine confers any protection in children aged below 2 years. Furthermore CVD103-HgR would not be expected to confer protection against V. cholerae O139 (Ryan et al., 2000).
The following US patent disclose different strains of Vibrio cholerae and vaccine derived therefrom: U.S. Pat. Nos. 4,264,737; 4,666,837; 4,882,278; 5,631,010; 5,882,653; 4,935,364; 5,098,998.